The transcriptome of Plasmodium vivax reveals divergence and diversity of transcriptional regulation in malaria parasites Zbynek Bozdech*, Sachel Mok*, Guangan Hu*, Mallika Imwong†, Anchalee Jaidee‡, Bruce Russell§, Hagai Ginsburg¶, Francois Nosten‡, Nicholas P. J. Day†, Nicholas J. White†, Jane M. Carltonʈ, and Peter R. Preiser*,** *School of Biological Sciences, Nanyang Technological University, Singapore 637551; †Wellcome Trust Mahidol University Oxford Tropical Medicine Research Programme and ‡Shoklo Malaria Research Unit, Faculty of Tropical Medicine Research, Mahidol University, Salaya, Nakhon Pathom 73170,Thailand; §Laboratory for Malaria Immunobiology, Singapore Immunology Network, Biopolis, Agency for Science, Technology and Research, Singapore 138632; ¶Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; and ʈDepartment of Medical Parasitology, New York University Langone Medical Center, New York, NY 10010 Communicated by Louis H. Miller, National Institutes of Health, Rockville, MD, August 18, 2008 (received for review May 14, 2008) Plasmodium vivax causes over 100 million clinical infections each year. to relapse periodicity and chloroquine sensitivity, which the mech- Primarily because of the lack of a suitable culture system, our under- anisms behind these differences are still unknown. standing of the biology of this parasite lags significantly behind that Investigations into the biology of P. vivax have been restricted by of the more deadly species P. falciparum. Here, we present the the lack of a continuous cultivation system. With advances of complete transcriptional profile throughout the 48-h intraerythro- sequencing technology, the P. vivax genome is now available (13), cytic cycle of three distinct P. vivax isolates. This approach identifies allowing the construction of a representative microarray. This strain specific patterns of expression for subsets of genes predicted to significant advance, coupled with the ability to mature ex vivo encode proteins associated with virulence and host pathogen inter- isolates, has opened the way to obtain a high-quality transcriptome actions. Comparison to P. falciparum revealed significant differences of the blood stages. This study aimed to provide a stage-specific in the expression of genes involved in crucial cellular functions that transcriptome of the intraerythrocytic developmental cycle (IDC) underpin the biological differences between the two parasite species. of P. vivax which can be compared with P. falciparum (6). Although These data provide insights into the biology of P. vivax and constitute an the IDC represents only a relatively small portion of the Plasmo- important resource for the development of therapeutic approaches. dium life cycle, close to two thirds of Plasmodium genes are expressed and transcriptionally regulated during this 48-h develop- comparative genomics ͉ Plasmodium falciparum ment (6, 7). Thus, characterizing the P. vivax IDC transcriptome will provide broad insights into the P. vivax biology and gene function- t is now increasingly recognized that P. vivax infections contribute alities of this parasite. In addition, three separate clinical isolates of Isignificantly to the burden of malaria (1, 2). In all endemic areas P. vivax were used to provide some indication into the magnitude except for Africa, P. vivax is often the dominant species, and at least of intraspecies IDC transcriptome variation [see supporting infor- 100 million cases are reported annually (2, 3). Although vivax mation (SI) Dataset S1, Dataset S2, and Dataset S3]. malaria is clinically less likely than P. falciparum to develop into a life threatening disease, it exerts a substantial toll on the individual’s Results and Discussion health and economic well being. The chronic, long-lasting nature of Transcriptional Regulation of P. vivax Genes During the Erythrocytic the infection contributes substantially to morbidity. Chronicity is Stage. To study the IDC transcriptome of P. vivax, we collected because of hypnozoites, dormant liver stages from which fresh three clinical isolates from acute vivax malaria patients before blood infection or relapses originate up to 2 years after the infectious bite (4). The presence of hypnozoites make infections by treatment on the Northwestern border of Thailand (Shoklo Malaria P. vivax difficult to cure radically and pose a serious obstacle to the Research Unit, Mahidol University, Mae Sot, Thailand). These control and eventual eradication of this parasite. synchronous, monoclonal, erythrocytic isolates were cultured ex The description of the P. falciparum genome (5) and staged vivo from the early ring stage to the schizont stage (see Table S1) erythrocytic transcriptome (6, 7) has provided an invaluable re- (12, 14). Transcriptional analysis using a genome-wide long oligo- source for the study of this important species. It would be of nucleotide microarray designed by the recently established algo- fundamental and practical interest to do the same for P. vivax rithm OligoRankPick (15) showed that during the P. vivax IDC, because there are important biological and clinical differences each gene is activated at a particular developmental stage analo- between this species and P. falciparum, whose basis is currently gous to P. falciparum (Fig. 1) (6). To evaluate the reproducibility unknown (8). For example, the presence of circulating mature and fidelity of the microarray results, one of the IDC transcriptome erythrocytic stages of P. vivax would suggest that multigene families was replicated in a dye swap experiment, and expression profiles for and processes implicated in antigenic variation and immune evasion 5 genes were verified by quantitative RT-PCR (Fig. S1). Similar to are quite different to P. falciparum, whose mature asexual red cell P. falciparum, the IDC transcriptome of P. vivax shows that func- stages generally sequester. Unlike P. falciparum, P. vivax has a selective preference for infecting reticulocytes (9), strongly suggest- ing an alternate red cell attachment invasion mechanism. In con- Author contributions: Z.B., M.I., F.N., N.P.J.D., N.J.W., and P.R.P. designed research; S.M., M.I., and A.J. performed research; G.H., M.I., B.R., F.N., and J.M.C. contributed new trast to the rigid, sticky and knobby P. falciparum infected red cell, reagents/analytic tools; Z.B., S.M., G.H., B.R., H.G., and P.R.P. analyzed data; and Z.B., B.R., P. vivax remodels the host-cell membranes to produce a highly and P.R.P. wrote the paper. deformable erythrocyte characterized by numerous caveola-vesicle The authors declare no conflict of interest. complexes (10–12). Finally, the kinetics of gametocyte production **To whom correspondence should be addressed at: School of Biological Science, Nanyang in P. vivax is also different than P. falciparum,withP. vivax Technological University, 60 Nanyang Drive, Singapore 637551. E-mail: [email protected]. gametocytes appearing much earlier and being relatively short lived This article contains supporting information online at www.pnas.org/cgi/content/full/ (8). Aside from these notable interspecies differences, there are a 0807404105/DCSupplemental. number of important phenotypic differences within P. vivax relating © 2008 by The National Academy of Sciences of the USA 16290–16295 ͉ PNAS ͉ October 21, 2008 ͉ vol. 105 ͉ no. 42 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807404105 Downloaded by guest on September 29, 2021 transition (Fig. 1B) can be divided into three broad classes: immune evasion, host-cell invasion, and functionally uncharacterized genes. The vir gene family is the largest gene family in P. vivax, members of which have been implicated in immune evasion (16). Of the 346 vir genes predicted in the P. vivax genome, at least 204 are transcribed during the IDC. Although distinct groups of vir genes are expressed at different IDC stages (Fig. 1B), no correlation was observed between the time of expression and the postulated phylogenic groups (16) (Fig. S2). Members of the pvtrag gene family that have also been linked with P. vivax immune evasion (17) show two distinct phases of transcription (Fig. 1B). These data suggest that during its IDC, P. vivax undergoes two ‘‘waves’’ of antigenic presentation. The first wave is initiated immediately after invasion by expression of a large proportion of the vir and pvtrag genes family (121 vir and 12 pvtrag), and the second wave is timed to schizogony during which another large, but nonoverlapping, group of both gene families (52 vir 13 pvtrag) are expressed. This second wave is potentially reflecting additional needs for antigenic presentation of the nonsequestering P. vivax parasite. This presentation of variant antigens is clearly different from P. falciparum, in which transcrip- tion of the majority of the antigenic gene families is silenced in the late stages (18–20). Expression during the schizont-ring transition was also detected for gene families that had undergone lineage- specific evolution. These include genes linked with host-cell inva- sion like merozoite surface protein 3 (msp3), msp7, and reticulocyte binding proteins (rbp) and two functionally uncharacterized gene families Pv-fam-e (RAD) and Pf-fam-h (PHIST) (Fig. S3). Based on these observations, it is likely that the new members of these gene families, as well as 133 ring- and 156 schizont-specific, nonsyntenic, hypothetical genes expressed during the IDC (Fig. 1B), represent important factors associated with the